Mu t an t p53 Forms a Complex wi t h Sp1 on H I V-LTR DNA Agustin Chicas, Patricia Molina, and Jill Bargonetti 1 Institute for Biomolecular Structure and Function, and Department of Biological Sciences, Hunter College and The Graduate Center, City University of New York, 695 Park Avenue, New York, New York 10021 Received November 7, 2000 Many mutants of p53 activate HIV-LTR driven tran- scription and promote HIV replication. The region of the HIV-LTR containing Sp1-binding sites is impor- tant for this effect. In this study we test the hypothesis that mutant p53 interacts with DNA-bound Sp1 and in this way can increase transcription from Sp1- dependent promoters. We have used the breast cancer cell line MDA-MB-468 that expresses endogenous mu- tant p53 His273 as our source of p53 protein. First, we demonstrated that this mutant p53 participates in ac- tivating transcription from the HIV-LTR by showing that HIV-LTR-directed transcription in MDA-MB-468 cells is inhibited in a dominant-negative manner by p53 Val135 . Using HIV-LTR DNA affinity chromatogra- phy, we detected coelution of p53 His273 and Sp1. We also demonstrated that this mutant p53 binds sequence specifically to the super consensus sequence (SCS) and that Sp1 coeluted with p53 His273 from a column contain- ing this site. These data indicate that p53 His273 can as- sociate with DNA-bound Sp1 suggesting that activated HIV-LTR transcription associated with mutant p53 oc- curs through a DNA driven multi-protein complex. © 2000 Academic Press Key Words: His 273-p53; DNA binding; transcription; Sp1. Mutation of the tumor suppressor p53 is one of the most common genetic alterations found in cancer cells (1, 2). The tumor suppressor activity of wild-type p53 is dependent on the sequence-specific DNA binding and transcriptional activation ability of the protein (3). Most tumor derived mutants of p53 are compromised in their ability to activate transcription of p53 respon- sive genes due to mutations in the central DNA- binding domain of the protein (4 – 6). Oncogenic mutants of p53 proteins not only lose their tumor suppressor activity but also gain oncogenic properties (7, 8). It has been proposed that one onco- genic property of mutant p53 may be due to the ability of mutant p53 to activate the transcription of genes that can enhance cell proliferation (9). Consistent with this, tumor-derived p53 mutants have been shown to activate transcription of a number of such promoters. The list includes, but is not limited to, epidermal grow factor receptor (egfr) (10), proliferating cell nuclear antigen (pcna) (11), the multi-drug resistant gene (mdr-1) (12), and c-myc (13). Additionally many mu- tant p53 proteins promote active HIV replication when introduced into cells latently infected by the virus (14). This correlates with the ability of these mutant p53 proteins to activate transcription of a reporter gene driven by the HIV-LTR (15, 16). Mutant p53 proteins may activate transcription of the HIV-LTR by cooperation with the transcription factor Sp1 as the Sp1-binding sites of the virus are required for full mutant p53 transactivation (15–17). Sp1 is a ubiquitous transcription factor that binds to specific GC rich DNA sequences called GC Boxes that are present in many eukaryotic housekeeping genes (18). Although Sp1 is known for its role in basal pro- moter activity, it has become evident that Sp1 can work together with other enhancer-binding proteins to synergistically activate the transcription of many dif- ferent genes (19). Some transcription factors coactivate transcription of Sp1 containing promoters through as- sociation with the Sp1 protein rather than by directly binding to the promoter (20). Wild-type p53 and Sp1 have been shown to physically associate with one an- other when TF-1 cells are treated by Granulocyte Mac- rophage Colony Stimulating Factor (21). A p53–Sp1 association is also induced in Jurkat cells when these cells are treated with TNF-alpha. In the latter case it has been shown that TNF-alpha causes p53 to shift into a mutant-like conformation as determined by a change in antibody-reactivity (16); the denatured con- formation of p53 is specifically recognized by the p53 antibody PAb240 (22). The Sp1–p53 complex has been suggested to mediate TNF-alpha-induced NF-kB- independent transactivation of the HIV-LTR (16). Since many tumor-derived mutants of p53 activate HIV-LTR directed transcription in the absence of mi- togen stimulation (15), these results suggest that such 1 To whom correspondence should be addressed. Fax: 212-772- 5227. E-mail: bargonetti@genectr.hunter.cuny.edu. Biochemical and Biophysical Research Communications 279, 383–390 (2000) doi:10.1006/bbrc.2000.3965, available online at http://www.idealibrary.com on 383 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.